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Closely Watched Study Fails to Find Arsenic in Microbial DNA

The debate over whether a bacterium can incorporate arsenic into its DNA just flared up again, with the posting yesterday of a paper refuting the idea on ArXiv, an electronic preprint archive primarily used by astronomers, mathematicians, and physicists. The controversy began in December 2010, when NASA astrobiology fellow Felisa Wolfe-Simon and colleagues described online in Science a microbe called GFAJ-1, which grew, albeit slowly, in the presence of arsenic, leading the authors to conclude the bacterium had taken up the toxic element and incorporated it into its cellular components. The report, amplified by a NASA press conference, quickly lit up the blogosphere and Twitter and led to the unprecedented publication of eight critical technical comments alongside the print version of the paper.

Wolfe-Simon and her colleagues agreed to make samples of GFAJ-1 available and now one vocal critic, Rosemary Redfield, a microbiologist at the University of British Columbia, Vancouver, in Canada, has grown the bacterium in the presence of arsenic and found no evidence of its uptake in the microbe's genetic material. "The data they have supports the conclusion there is no arsenic in the DNA," comments Michael Bartlett, a chemist at the University of Georgia, Athens, who is an expert in mass spectrometry of DNA, RNA, and related molecules.

Redfield, who chronicled every twist and turn of her experiments on her blog, and some other critics of the original paper are satisfied this resolves the matter and do not plan follow-up experiments. "We can do fancier analyses that push the limits of detection down, but I think the burden of proof is back on the authors," she says. "They are going to have to provide some better data than they did in their paper."

But Wolfe-Simon and her colleagues say the work on arsenic-based life is just beginning. They told ScienceInsider that they will not comment on the details of Redfield's work until it has been peer reviewed and published. They emphasize that the key point of their paper was that the microbe was able to use arsenic to grow, despite its toxicity, and that they only suggested arsenic was in the DNA. "What this is about is refuting an extreme interpretation of the paper," says John Tainer, a biochemist who was not an author on the original paper but is now working with Wolfe-Simon at the Lawrence Berkeley National Laboratory in California. "I would call it more of an attack than a paper. It aims to shut the door on additional research."

At the heart of the Wolfe-Simon paper was the finding that when starved of phosphorus, an element vital to life and a key component of DNA, and when provided with ample arsenic, GFAJ-1 was able to grow and use the arsenic instead of phosphorus. Analytical tests indicated that arsenic might have been used to make DNA. (The current assertion by Wolfe-Simon and her colleagues that the original paper did not specifically claim arsenic was in GFAJ-1's DNA has already drawn criticism and a detailed rebuttal from one scientist who had previously defended Wolfe-Simon.)

Yet Redfield and others argued that it was impossible for arsenic to be part of DNA and worried that the arsenic found associated with GFAJ-1 was contamination and not part of any of its biomolecules. They also suggested that the little bit of phosphorus already in the bacterial growth media would have been enough to fuel the growth attributed to arsenic.

When Redfield grew the microbe in her own lab, she got no growth at first. Then by adding an amount of phosphorus that she estimated to be equivalent to phosphorus found in the media used by Wolfe-Simon's team, Redfield saw growth rates equivalent to what Wolfe-Simon saw. Growth was similar in samples with and without added arsenic. Thus Redfield concludes that the minimal phosphorus, not the added arsenic, fueled the growth recorded by Wolfe-Simon.

Redfield has been blogging her day to day research for about 6 years, and she continued by posting regular updates on her attempts to grow GFAJ-1, eventually asking for collaborators on the project. Princeton University's Leonid Kruglyak answered Redfield's call for help as he has a mass spectrometry system able to analyze the constituents of DNA. Princeton graduate student Marshall Louis Reaves subjected isolated GFAJ-1 DNA to liquid chromatography-mass spectrometry and found no signs of arsenic in the DNA.

"This lab knows how to look at and detect these types of molecules," says Patrick A. Limbach, a chemist who studies mass spectrometry of nucleic acids at the University of Cincinnati. Based on the new data, "it's very clear there is no arsenate associated in the DNA backbones."

Redfield has suggested that the arsenic Wolfe-Simon's group claimed was in the microbe's DNA was contaminating arsenic that wasn't washed away when the DNA was purified. Redfield and her colleagues, says Bartlett, "have gone to significant lengths to make sure they remove any residual arsenic that might be associated with the [DNA]. The [Wolfe-Simon] paper did not."

However, Bartlett does take issue with how sensitive Redfield says her tests were to the presence of arsenic. He doesn't not think the limit of detection was 50-fold lower than what Wolfe-Simon found, as Redfield claims. There still could be some arsenic in the bacterium's biomolecules, he says, though not nearly as much as the Wolfe-Simon team originally reported finding.

Tainer and Wolfe-Simon don't understand why Redfield didn't see arsenic-stimulated growth, but say the original paper stands up. "If there is no arsenic in DNA, [then] where is the arsenic going? That's the cool scientific question," says Tainer, one that he and Wolfe-Simon are pursuing by looking in the ribosome and elsewhere in the cell. And in her paper, Redfield also leaves open that possibility, suggesting that arsenic may be taken up by other molecules, just not DNA.

While the new work has not yet been formally reviewed by a journal—Redfield says it has been submitted to Science—other scientists have regularly commented and offered advice on her blog posts. As for the use of ArXiv, Kruglyak notes that posting papers on a preprint server has become commonplace in the physical sciences, but not in biology. Redfield says that Science's editors assured her that putting the paper on ArXiv would not jeopardize the paper's chance for publication there. Already several comments have come in with suggestions on improving the work. "I think it's a very positive thing," Kruglyak adds.

Not everyone agrees with Redfield's preprint and blogging approach, however. "To me it sort of looks like publicity hunting by Redfield," says Bartlett. "It's not the way I'd like to do science."